Functional distribution of epithelial sodium channels and water channels in the amphibian skin

为了定位上皮钠通道（ENaC），我们建立了针对从A6肾细胞系中克隆的爪蟾ENaC (α xenac) α亚基胞外环肽的多克隆抗体。在荒漠蟾蜍（Bufo alvarius）中，α ENaC成功定位于颗粒细胞层最浅层细胞的顶端细胞膜。接下来，我们使用了针对从树蛙腹侧皮肤中克隆的水通道蛋白（aquaporin-2: AQP-h2）的c端肽的抗体。该抗体识别30.0 kd蛋白，主要存在于B.alvarius的腹腔皮肤中。在皮肤的其他区域，如胸部和背部皮肤，没有观察到免疫反应，这与沙漠蟾蜍利用盆腔皮肤的吸水行为是一致的。免疫反应性在颗粒层上层强烈，并局限于主细胞的顶端细胞膜。腹侧皮肤暴露于抗利尿激素（AVT, 10^<-8>M）可促进根尖膜更强烈的免疫反应性。结果说明沙漠蟾蜍可以检测到吸水底物中的钠离子。

To localize the epithelial sodium channel (ENaC) we raised a polyclonal antibody against a peptide in the extracellular loop of the α subunit of Xenopus ENaC (αxENaC) that had been cloned from A6 kidney cell line. In desert toads, Bufo alvarius, the α ENaC was successfully localized in the apical cell membrane of the most superficial cells in the granulosum cell layer. Next, we used an antibody against, the C-terminal peptide of a water channel protein (aquaporin-2 : AQP-h2) that had been cloned from the ventral skin of the tree frog, Hyla japonica. The antibody recognized a 30.0 kD-protein and immunostained predominantly in the abdominal pelvic skin of B.alvarius. No immunoreactivity was seen in the other area of the skin such as the pectoral and dorsal skins, which is consistent to a water absorption behavior of the desert toads that utilize the pelvic skin. The immunoreactivity was intense in the upper layer of the stratum granulosum and was localized to the apical cell membrane of the principal cells. Exposure of the ventral skin to anti-diuretic hormone (AVT, 10^<-8>M) facilitated more intense immunoreactivity in the apical membrane. Results explain that the desert toad can detect sodium ions in the substrate of water absorption.